Recipient Organization
NORTH CAROLINA A&T STATE UNIV
1601 EAST MARKET STREET
GREENSBORO,NC 27411
Performing Department
Sponsored Programs
Non Technical Summary
Metabolic syndrome has reached epidemic proportions and remains the leading cause of preventable diseases including type 2 diabetes and heart diseases. An impressive body of evidence supports the notion that increasing consumption of whole-grain and/or cereal bran is associated with lower risk of obesity, diabetes, and heart diseases. The bran fraction of the whole-grain contains important bioactive phytochemicals and is the major source of cereal fiber. It is still unclear whether the observed beneficial health effect of cereal bran is due to phytochemicals or fiber or the combination of phytochemicals and fiber. Wheat bran is one of the most important dietary cereal brans and its consumption has been found to decrease the risk of many different chronic diseases. We recently studied the chemical profile of wheat bran and found that it contains three major types of bioactive components: alkylresorcinols, sphingolipids, and sterols and sterol ferulates. Studies have shown that these major phytochemicals may have a protective effect against the development of metabolic syndrome. Based on the literature and our preliminary data, our hypothesis is that the effect of wheat bran on metabolic syndrome resides in the additive and/or synergistic interactions among various bioactive components. The goal of this project is to characterize the roles of phytochemicals and fiber in wheat bran in the prevention of high-fat diet-induced metabolic syndrome. The present study will provide important information on the in vivo effectiveness of the phytochemicals in WB in the development of metabolic syndrome.
Animal Health Component
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Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Goals / Objectives
The project'sspecific objectives are as follows: Objective 1. To determine the effects of the two fractions of wheat bran, phytochemicals and fiber, on metabolic syndrome in high-fat-fed mice. Objective 2. To elucidate the mechanisms of inhibition of body weight gain and type 2 diabetes by wheat bran and its two major fractions.
Project Methods
Objective 1 -- To determine the effects of the two fractions of wheat bran, phytochemicals and fiber, on metabolic syndrome in high-fat-fed mice. Experimental design and methods 3.1.1 Preparation of wheat bran phytochemicals and wheat bran fiber. Following scientistsprevious method [34], raw WB (5 kg) will be ground and extracted four times with 95% ethanol at 40 °C (each time 5 h). After evaporation, the residue will be considered as the WB phytochemicals (about 500 g). The ethanol insoluble fraction will be dried and considered as the WB fiber (4.5 kg). The WB phytochemicals and WB fiber as well as the raw WB will be sent to Research Diet Inc. (NJ) to make all the required diets. 3.1.2 Animal treatment and sample collections C57BL/6J mice (male, 6 wks of age) will be divided into 8 groups (15 mice in each group, see Statistical Analysis Section) in the following design: 1. Negative control group on low fat diet (10% energy as fat) 5. WB phytochemicals (0.5%) in high fat diet 2. Control group on high fat diet (60% energy as fat) 6. WB phytochemicals (1.0%) in high fat diet 3. WB (5%) in high fat diet 7. WB fiber (4.5%) in high fat diet 4. WB (10%) in high fat diet 8. WB fiber (9.0%) in high fat diet Food and fluid intake and body weight will be measured weekly. Signs of abnormality and possible toxicity will be monitored. Fasting blood glucose will be measured at 0, 5, 10, 12, 14, and 16 weeks of treatment. Food will be removed 8 h prior to blood glucose measurements, and the cage bedding will be changed to minimize the interference from coprophagy. Blood will be collected from the tail vein, and glucose levels will be measured with a One Touch® Ultra® 2 glucose monitor. Mice will be food-deprived for 8 h and sacrificed after 16 weeks of treatment. Whole blood will be obtained by cardiac puncture. Liver, omental fat, and retroperitoneal fat will be harvested, rinsed, and weighed. Plasma will be isolated by centrifugation at 5000 x g for 15 min. A liver will be considered fatty based on altered the percentage of fatty livers will be recorded in each group. In addition, fecal samples will be collected at 0, 5, 10, 12, 14, and 16 weeks of treatment for measuring fecal lipid levels. All samples will be stored at -80 ºC. 3.1.3 Biochemical analysis of plasma samples Plasma levels of fasting insulin, total cholesterol, HDL, and triglycerides will be measured. Insulin levels will be measured by ELISA (Millipore, Billerica, MA, USA) following the manufacturer's protocol. Levels of total cholesterol, HDL, and triglycerides will be determined using a standard enzymatic assay (Pointe Scientific, Canton, MI, USA). The hepatic triglyceride content will also be measured using similar method. The free fatty acid levels in plasma will be measured using an ELISA kit (Cayman Chemical, Ann Arbor, MI, USA). 3.1.4 Quantify the levels of the major WB phytochemicals in plasma Scientists will modify established LC/MS method to analyze the plasma levels of the major WB phytochemicals. Plasma sample (50 µL) will be prepared in the presence of b-glucuronidase (250 U) and sulfatase (1 U) for 45 min at 37°C and then extracted twice with ethyl acetate. After evaporation, the ethyl acetate fraction will be dissolved in methanol for LC/MS analysis. Duplicate samples will be prepared in the absence of glucuronidase/sulfatase to determine the level of unconjugated compounds in the sample. It has been reported that WB fiber can be metabolized by gut flora to generate short-chain fatty acids, such as butyric acid and the ferulic acid bound to fiber can be hydrolyzed by gut flora in vivo. Therefore, we will also quantify the levels of butyric acid and ferulic acid in WB and WB fiber treated mice. The PD has extensive experience in the analysis of dietary compounds and their metabolites using LC/MS [35-41]. 3.2. Objective 2-- To elucidate the mechanisms of inhibition of body weight gain and type 2 diabetes by wheat bran and its two major fractions. Experimental design and methods 3.2.1 Fecal lipids If scientists observe the decrease of body weight and body fat gains, fecal lipid levels will bemeasured to determine whether WB and its active components inhibit lipid absorption or increases fat oxidation. Feces collected at 0, 5, 10, 12, 14, and 16 weeks of treatment will be weighed (1 g of each sample), added to 4 mL deionized water, and allowed to sit at 4 °C overnight. Following vortexing, lipids will be extracted with methanol: chloroform (2:1, v:v) using a previously described method [42]. The lipophilic layer from the extraction will be collected and dried under vacuum. Total lipids will be measured gravimetrically. 3.2.2 Plasma inflammatory markers, Plasma monocyte chemoattractant protein (MCP)-1, tumor necrosis factor (TNF)-α, and C-reactive protein will be determined using separate ELISA kits (R&D Systems for MCP-1, Invitrogen for TNF-α, and Alpco Diagnostics for C-reactive protein). 3.2.3 Plasma leptin and adiponectin levels and Adipose tissue adiponectin and (PPAR)-γ levels Plasma leptin and adiponectin levels will be measured using commercial ELISA kits provided by B-Bridge International, Inc. and Linco Research, respectively. Levels of adiponectin and PPAR-γ in adipose tissue will be determined using separate ELISA kits (Alpco Diagnostics for adiponectin and ELAb UNCNLIFE for PPAR-γ).